Gene cluster for rabelomycin biosynthesis and its use to generate compounds for drug screening

ABSTRACT

This invention relates to the gene cluster for angucycline biosynthesis, derived from Streptomyces, and use of the genes therein to obtain antibiotics for drug screening.

This invention relates to the gene cluster for angucycline biosynthesis,derived from Streptomyces, and use of the genes therein to obtainantibiotics for drug screening.

BACKGROUND OF THE INVENTION

Tetracyclic aromatic polyketides known as angucyclines were firstisolated from bacterial cultures over thirty years ago. The angucyclinegroup of antibiotics has become a rapidly growing group of bioactivenatural products, whose members are discovered by diverse screeningmethods such as antibacterial, antitumor and chemical screens.

These compounds are biosynthetized in microbes by polyketide pathway bytype II polyketide synthase. The polyketide is folded in a mannercharacteristic to angucyclines: the fourth ring is orientated in anangular fashion, as described by the name ‘angucycline’. The aglyconeformed is subsequently modified by diverse reactions, such as oxidation,hydroxylation and glycosylation at various positions, to give a varietyof structures. Furthermore, chemical synthesis to create angucyclinesfor drug discovery purposes has been described. Biosynthesis geneclusters for a few angucycline antibiotics have been cloned andpartially characterized; the clusters for urdamycin from Streptomycesfradiae, landomycin from S. cyanogenus S136, jadomycin from S.venezuelae ISP5230, and pradimicin from Actinomadura verrucosospora(Decker et al., 1995, Westrich et al., 1999, Han et al., 1994, Dairi etal., 1999, respectively). The clusters for kinamycin from S.murayamaensis (Gould et al., 1998), tetrangulol and tetrangomycin fromS. rimosus and PD 116740 from the Streptomyces strain WP 4669 (Hong etal., 1997) have been cloned and expressed in heterologous hosts. Thegene cluster for pradimicin is disclosed in an international patentapplication of Oki et al. (WO 98/11230).

The angucycline antibiotics exhibit diverse bioactivities. Besides anantitumor activity, some of the angucyclines act as enzyme inhibitors,potent inhibitors of blood platelet aggregation, and most of themexhibit antimicrobial activity. In vivo cytostatic activities werereported for the kerriamycins and antibiotic SS-228Y, which can prolongthe survival periods of mice inoculated with Erlich ascites tumors.Vineomycins exhibit antitumor activity against Sarcoma 180 solid tumorin mice. Remarkably, some of the members of the angucycline group havebeen described as inhibiting the growth of cell lines resistant tovarious cytostatics in market.

For the literature of the angucycline group concerning chemicalsynthesis, biosynthesis, bioactivites and the molecular structures, seethe reviews by Krohn and Rohr (1997) and by Rohr and Thiericke (1992)and the references therein.

SUMMARY OF THE INVENTION

The present invention concerns a gene cluster derived from Streptomycesbacteria, especially that of the strain Streptomyces sp. H021, which isinvolved in angucycline biosynthesis. The strain used for gene cloningfailed to produce angucyclines in several culture conditions tested inour laboratory. However, expressing a DNA fragment of said cluster in S.lividans or in S. coelicolor, rabelomycin, 5-OH-rabelomycin, and a novelcompound, 11-OH-rabelomycin, were obtained. These compounds are membersof the angucycline group. Furthermore, when the cluster was introducedinto the Streptomyces hosts S. argillaceus and S. galilaeus, theygenerated a novel compound, 9-O-methyl-rabelomycin, and a prior knowncompound, ε-rhodomycinone, respectively.

Consequently, a primary object of the invention is the DNA fragmentwhich is the gene cluster for rabelomycin biosynthetic pathway ofStreptomyces bacteria, which fragment is included in two 9.5 kb flankedPstI fragments of Streptomyces genome. Further objects of the inventionare a recombinant DNA comprising said DNA fragment, and a process forproduction of hybrid compounds, specifically hybrid anthracyclines andaromatic polyketides, by transferring the DNA fragment of the inventioninto a Streptomyces host to obtain angucyclines for drug screening.

DETAILED DESCRIPTION OF THE INVENTION

The experimental procedures used in the present invention are methodsconventional in the art. The techniques not explained in detail here aregiven in the manuals by Hopwood el al. “Genetic manipulation ofStreptomyces: a laboratory manual”, The John Innes Foundation, Norwich(1985) and by Sambrook et al. (1989) “Molecular cloning: a laboratorymanual”. The publications, patents and patent applications cited hereinare given in the reference list in their entirety, and they areincorporated herein by reference.

The present invention concerns particularly the gene cluster for theangucycline biosynthesis (11P2), causing the production of rabelomycinand its derivatives in S. lividans, a non-producer of angucyclines. Inspecific, the invention concerns the use of the genes for rabelomycinbiosynthesis to generate hybrid products modified in several positionswhen expressed in S. lividans, or in S. argillaceus, a producer ofmithramycin. Furthermore, the invention concerns the gene fragment11P23, that contains genes involved in sugar biosynthesis.

The biosynthetic genes for angucyclines can be isolated fromStreptomyces spp., particularly from such strains which give a positivehybridization signal by a short fragment of ketosynthase I (KS I) forrabelomycin biosynthesis. Since these genes were silent in the donorstrain Streptomyces sp. H021 used in our experiments, it will beappreciated that as a donor any actinomycete, especially a streptomycetebacterium can be used, obtained by screening with DNA-fingerprintingtechniques with the primers similar to rabelomycin KS I gene.

A bacterial strain carrying the genes for rabelomycin can be isolatedfrom a soil sample by any conventional screening method, but especiallyDNA fingerprinting of polyketide (Type II) is suitable. The primers forDNA fingerprinting are degenerated nucleotide oligomers sharing thesequences 5′-TSGCSTGCTTCGAYGSATC-3′ (SEQ ID NO:21) and5′-TGGAANCCGCCGAABCCGCT-3′ (SEQ ID NO:22). The bacterial strain thatgave a DNA fragment similar to angucyclines in PCR reaction, using theprimers as described, was used to deliver DNA for the construction ofthe gene library.

Genomic DNA of a Streptomyces strain containing the genes forrabelomycin biosynthesis is used in preparing a gene library. Suitablegene fragments for cloning may be obtained by any frequently digestingrestriction enzyme. Typically Sau3AI is used. The isolated fragments canbe inserted by ligation in any Escherichia coli vector, such as aplasmid, a phagemid, a phage, or a cosmid, though a cosmid vector ispreferred, since it enables cloning of large DNA fragments. A cosmidvector, such as pFD666 (ATCC Number 77286) is suitable for this purpose,as it enables cloning of fragments of about 40 kb. BamHI site of pFD666,giving sticky ends to the Sau3AI fragments, may be used for cloning. Topackage a ligation mixture containing recombinant cosmids in phageparticles, commercially available kits may be used. Several E. colistrains can be used for infection by the recombinant cosmids packaged,and a suitable one is e.g. E. coli XL1 Blue MRF′, deficient in severalrestriction systems.

Using E. coli as a host strain for a gene library, hybridization is anadvantageous screening strategy. The probe for hybridization may be anyknown fragment derived from the rabelomycin gene cluster but a shortfragment of 613 nt, prepared by multiplying a region from ketosynthase Iwith degenerated primers, is preferred. Colonies for the gene libraryare transferred to membranes for filter hybridization, and nylonmembranes are typically used. Any method for detection for hybridizationmay be used but, in particular, the DIG System (Boehringer Mannheim,GmbH, Germany) is useful. Since the probe is homologous to thehybridized DNA, it is preferable to carry out stringent washes ofhybridization at 68° C. in a low salt concentration, according toBoehringer Mannheim's manual, DIG System User's Guide for FilterHybridization. At least 80%, preferably 90%, homology is suggested to beneeded for a DNA fragment to be bound to a probe in the conditions usedfor washes.

Using this protocol, two clones out of about 1000 gave positive signalsand were picked up for DNA isolation. Restriction mapping is anappropriate technique for characterizing the clones. The positive clonesmay be digested with convenient restriction enzymes to demonstrate thephysical linkage map of the DNA fragments. We designated the positiveclones obtained as pFDH0211.1 and pFDH0216.1. In expression studies wepreferred to use pIJ486, a high copy number Streptomyces plasmid.However, any plasmid which is able to stably replicate in Streptomycesmay be used. The clone pFDH0211.1 was transferred into S. lividans TK24as two PstI-fragments inserted into pIJ486. The two recombinant plasmidsobtained were designated as pS11P2 and pS11P23 containing 9.5 kbfragments from H021 genomic DNA. These were further introduced intoother Streptomyces strains by protoplast transformation.

In TK24 the plasmid pS11P2 caused the production of rabelomycin and its5-OH and 11-OH derivatives. A further introduction into S. argillaceuscaused the production of 9-O-methylrabelomycin. In addition, whenexpressed in S. galilaeus H039, which producesaklavinone-rhodinose-rhodinose, the plasmid generates the production of11-OH-akla-vinone, also called ε-rhodomycinone, with correspondingsugars, suggesting that 11-hydroxylation activity is caused by a geneincluded in pS11P2. The plasmid pS11P23 caused the production of typicalaclacinomycins in S. galilaeus H075, which endogenously producesaklavinone-rhodosamine-deoxyfucose-deoxyfucose. The variety of themodifications in the Streptomyces strains used as hosts give promisingusefulness of the genes for combinatorial biosynthesis, to create novelcompounds and new chemical structures for drug discovery.

The sequence analysis can be made by any computer-based program, such asGCG (Madison, Wis., USA) package. Sequencing of the two flankingfragments, 11P2 and 11P23, used for cloning, consisting of 19016 bp,revealed 17 complete ORFs.

According to the present invention the putative gene functions asdeduced from the sequence homologies of those available in gene banksare: the orfs A, B and C code for minimal polyketide synthase (minPKS),ketosynthase I and II (KSI and KSII) and acyl carrier protein (ACP),respectively; orfD codes for polyketide ketoreductase; orfs E and M codefor oxygenases; orfs F and L code for polyketide cyclases; orfs V and Ocode for reductases; orfH codes for dTDP-glucose-4,6-dehydratase; orfQcodes for NDP-hexose-3-dehydroxylase; orfs (partial) codes forNDP-hexose-2,3-dehydratase; orfR codes for 4-ketohexose reductase; orfR1(partial) codes for a regulatory gene; orfJ codes for a transporterinvolved in resistance; orfl codes for protein of unknown function andorf2 codes for an oxidoreductase (see Table 1).

Streptomyces strains, in particular S. lividans, S. argillaceus and S.galilaeus, carrying the recombinant plasmids, are cultivated in mediawhich enable antibiotic production. The compounds, rabelomycin and itsderivatives, aclacinomycin and ε-rhodomycinone, are extracted withorganic solvents from the culture broth, and the compounds are separatedand purified using chromatographic techniques.

According to this invention the strain S. lividans TK24 carrying theplasmid pS11P2, and designated as TK24/pS11P2, produces rabelomycin,5-OH-rabelomycin and 11-hydroxy-rabelomycin in El medium, supplementedwith thiostrepton to give selection pressure for the plasmid containingstrains. The strain S. lividans TK24/pS11P2 and the strain TK24/pS11P23,carrying the plasmid containing the flanking region to 11P2, weredeposited according to the Budapest Treaty at Deutsche Sammlung vonMikroorganismen und Zelikulturen GmbH (DSMZ), Mascheroder Weg 1b,D-38124 Braunschweig, Germany on 13 Mar. 2001 with the accession numbersDSM 14172 and DSM 14173, respectively.

Any DNA fragment of the invention subcloned from a 19 kb rabelomycinbiosynthesis region can be inserted into a vector replicating inStreptomyces, and the products may be obtained by fermentation of thestrains carrying the plasmids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. shows the structures of rabelomycin (1), 9-O-methyl-rabelomycin(2), 5-OH-rabelomycin (3), 11-OH-rabelomycin (4), 19-methyl-SEK15 (5)and ε-rhodomycinone (6). The ring numbering used is also given.

FIG. 2 shows the gene cluster (11P232) of the invention. The PstIfragment from 1 to 9652 is the fragment 11P23 for complementation of themutant H075, and the fragment from 9647 to 19016 is the fragment 11P2for rabelomycin biosynthesis.

Examples to further illustrate the invention are given hereafter.

EXPERIMENTAL

Materials Used

Restriction enzymes used were purchased from Promega (Madison, Wis.,USA) or Boehringer Mannheim (Germany), alkaline phosphatase fromBoehringer Mannheim, and used according to the manufacturers'instructions. Proteinase K was purchased from Promega (Madison, Wis.,USA) and lysozyme from Sigma (St. Louis, Mich., USA). Hybond™-N nylonmembranes used in hybridization were purchased from Amersham(Buckinghamshire, England), DIG DNA Labelling Kit and DIG LuminescentDetection Kit from Boehringer Mannheim. Qiaquick Gel Extraction Kit fromQiagen (Hilden, Germany) was used for isolating DNA from agarose.Templates for sequencing were prepared using Template Generation SystemF-700 (Finnzymes, Finland) and the DNA sequencing was performed usingthe automatic ABI DNA sequenator (Perkin-Elmer) according to themanufacturer's instructions.

Bacterial Strains and Their Use

Escherichia coli XL1 Blue MRF′ (Stratagene, La Jolla, Calif.) was usedfor cloning. Streptomyces sp. H021 was isolated from a soil samplecollected from Turku, Finland and was studied due to the polyketideDNA-fingerprints obtained by the course of our genetical based screeningfor polyketide producers. The gene cluster of rabelomycin biosynthesiswas cloned from this strain.

The host strains to express the genes cloned were:

-   -   Streptomyces lividans TK24 (U.S. Pat. No. 5,986,077). This        strain was also used as a primary host to clone DNA propagated        in E. coli.    -   Streptomyces galilaeus H075, DSM 11638, (FI 105554 B) produces        aklavinone-rhodos-amine-2-deoxyfucose-2-deoxyfucose.    -   Streptomyces galilaeus H039 (Ylihonko et al. 1994) produces        aklavinone-rhodinose-rhodinose-rhodinose.    -   Streptomyces argillaceus ATCC 12956 produces mithramycin.        Plasmids

E. coli—Streptomyces shuttle cosmid pFD666 (ATCC 77286) was used forcloning the chromosomal DNA. E. coli cloning vector and pUC19 was usedfor making the sub-clones.

pIJ486 is a high copy plasmid vector provided by prof Sir David Hopwood,John Innes Centre, UK (Ward et al., 1986). To clone the probe TOPO TACloning Kit (Invitrogen, USA) was used according to the manufacturer'sinstructions.

Nutrient Media and Solutions

For cultivation of the strain H021 for total DNA isolation TSB mediumwas used. Lysozyme solution (0.3 M sucrose, 25 mM Tris, pH 8, and 25mMEDTA, pH 8) was used in the isolation of total DNA. TE buffer (10 mMTris, pH 8.0 and 1 mM EDTA) was used to dissolve DNA.

Tryptone Soya Broth (TSB)

Per litre: Oxoid Tryptone Soya Broth powder 30 g.

ISP4

Bacto ISP-medium 4, Difco; 37 g/l.

E1 Per liter in tap water: glucose 20 g  soluble starch 20 g  Farmamedia5 g Yeast extract 2.5 g   K₂HPO₄.3H₂O 1.3 g   MgSO₄.7H₂O 1 g NaCl 3 gCaCO₃ 3 g

pH adjusted to 7.4 before autoclaving

General Methods

Polyketide metabolites were detected by TLC (Kieselgel 60 F₂₅₄ glassplates) and HPLC on a Hewlett Packard instrument (1100 series) using aZorbax column (SB-C1 8, 3 μm, 4.6×150 mm) and gradient elution with amixture of MeCN—H₂O—HCO₂H (30:70:1).

NMR spectra were acquired on a JEOL JNM-GX 400 spectrometer equippedwith either a 5 mm normal configuration CH probe or a 5 mm inverse HXprobe operating at 400 MHz for ¹H and 100 MHz for ¹³C. The spectra wererun at 26° C. in the solvents indicated in Tables 2 to 4, and both ¹³Cand ¹H were referenced internally to TMS, assigned as 0 ppm. Electronimpact mass spectrometry spectra were taken on a VG Analytical Organicmass spectrometer 7070 E.

ISP4 plates supplemented with thiostrepton (50 μg/ml) were used tomaintain the plasmid carrying cultures.

EXAMPLE 1 Cloning the Gene Cluster for Rabelomycin Biosynthesis

1.1 Cosmid Library

For isolation of total DNA, the strain H021 was grown for three days in50 ml of TSB medium supplemented with 0.5% glycine. The cells wereharvested by centrifuging for 15 min at 3900×g in 12 ml Falcon tubes,and the cells were stored at −20° C. Cells from a 12 ml sample of theculture were used to isolate the DNA. 5 ml of lysozyme solutioncontaining 5 mg/ml lysozyme was added onto the cells, and incubated for20 min at 37° C. 500 μl of 10% SDS containing 1 mg of proteinase K wasadded onto the cells, and incubated for 90 min at 62° C. The sample waschilled on ice and 600 μl of 3M NaAc, pH 5.8 was added, and the mixturewas extracted with equilibrated phenol (Sigma). The phases wereseparated by centrifuging at 1400×g for 10 min. The DNA was precipitatedfrom the water phase with an equal volume of isopropanol, collected byspooling with a glass rod and washed by dipping into 70% ethanol, airdried and dissolved in 500 μl of TE-buffer.

The chromosomal DNA was partially digested with Sau3AI. The DNAfragments were separated by agarose gel electrophoresis and thefragments of 30 to 50 kb were cut from the 0.3% low gelling temperatureSeaPlaque® agarose. The DNA bands were isolated from the gel by heatingto 65° C., extracting with an equal volume of equilibrated phenol andthe phases were separated by centrifuging for 15 min at 2500×g. Thephenol phase was extracted with TE buffer, centrifuged and the waterphases were pooled. The DNA was precipitated by adding 0.1 volume ofNaAc, pH 5.8 and 2 volumes of ethanol at −20° C. for 30 min, centrifugedfor 30 min at 15 000 rpm in Sorvall RC5C centrifuge, using SS-34 rotorwith adapters for 10 ml tubes. The pellet was air dried and dissolved in20 μl of TE buffer. The isolated fragments were ligated to pFD666 cosmidvector digested with BamHI and dephosphorylated. The DNA was packed intophage particles and infected to E. coli using Gigapack® III XL PackingExtract Kit according to the manufacturer's instructions.

1.2 Identification of the Clones by Hybridization

The infected cells were grown on LB plates containing 50 μg/ml kanamycinand transferred to Hybond™-N nylon membranes (Amersham). DNA wasattached to membranes according to the protocol described in BoehringerMannheims manual “The DIG System User's Guide for Filter Hybridization”.The probe used to screen the colonies for the biosynthesis cluster wasprepared by multiplying a part of the ketosynthase gene with degeneratedprimers as described by Metsä-Ketelä et al. (1999), and cloned usingTOPO TA Cloning Kit (Invitrogen, USA). The plasmid carrying the probewas digested with EcoRI and the fragment was separated from the vectorby agarose gel electrophoresis and isolated from the gel using QiaquickGel Extraction Kit (Qiagen). The probe was labelled by digoxygeninaccording to Boehringer Mannheim's manual “The DIG System User's Guidefor Filter Hybridization”. Approximately 1000 colonies were screened byhybridization at 68° C., using the probe described. Positive colonieswere detected using DIG Luminescent Detection Kit (Boehringer Mannheim).Two colonies gave a positive signal. These clones were designated aspFDH0211.1 and pFDH0216.1. Cosmids from the positive clones wereisolated from a 5 ml culture by alkaline lysis method. Restrictionanalysis showed that the cloned fragmens overlapped each other,representing at least 50 kb of the continuous DNA.

1.3 Subcloning the Fragments for Sequencing

The clone pFDH0211.1 was digested with PstI, and two fragments of about9.5 kb were isolated and ligated to pUC19 that had been digested withPstI and dephosphorylated. These two fragments are located next to eachother in the H021 genome. The clones were named as p11P2 and p11P23, andthey were used as templates for sequencing, using Template GenerationSystem F-700 (Finnzymes, Finland). A subclone partially overlapping thefragments 11P2 and 11P23 that was prepared from pFDH0211.1 was alsosequenced, and this sequence confirmed that the fragments 11P2 and 11P23are located next to each other.

E. coli XL1 Blue MRF′ cells were cultivated overnight at 37° C. in 5 mlof LB-medium, supplemented with 50 μg/ml of kanamycin. For sequencingreactions the plasmids were isolated using alkali lysis method describedby Sambrook et al. (1989), and purified using Qiaquick Gel ExtractionKit from Qiagen, or the plasmids were isolated using Wizard PlusMinipreps DNA Purification System kit (Promega) according to themanufacturer's instructions. DNA sequencing was performed using theautomatic ABI DNA sequencer (Perkin-Elmer) according to themanufacturer's instructions.

1.4 Sequence Analysis and the Deduced Functions of the Genes

Sequence analyses were effected using the GCG sequence analysis softwarepackage (Version 8; Genetics Computer Group, Madison, Wis., USA). Thetranslation table was modified to accept also GTG as a start codon.Codon usage was analysed using published data (Wright and Bibb, 1992).

According to the CODONPREFERENCE program the sequenced DNA fragmentcontained 17 complete open reading frames (ORFs), as well as one 3′ endand one 5′ end of two other ORFs. The functions of the genes wereconcluded by comparing the amino acid sequences translated from theirbase sequences to the known sequences in data banks. The results areshown in the following Table 1 referring to the sequence data given inthe application. TABLE 1 Gene Position Homology %/ Accession Putativefunction product (SEQ ID NO) Similarity % number Polyketide minPKS KSIOrfA 13907-15142 UrdA (80/88) CAA60569 synthesis compl (2) KSII OrfB12681-13910 S. venezuelae AAB36563 compl (3) chain length determinant(71/80) ACP OrfC 12421-12684 S. venezuelae AAB36564 compl (4) acylcarrier protein (61/71) ketoreductase OrfD 11570-12352 S. venezuelaeAAB36565 compl (5) ketoreductase (80/90) oxygenase II OrfE 15586-17055UrdE (68/77) CAA60567 compl (6) cyclase OrfF 15208-15537 LanF (77/85)AAD13535 compl (7) cyclase OrfL 10557-11504 gris ORF4 (72/82) E55587compl (8) oxygenase I OrfM 9014-10555 LanM (62/73) AAD13541 compl (9)reductase I OrfV 8178-8939 UrdM (73/83) AAF00206 compl (10) reductase IIOrfO 4854-5438 LanO (63/72) AAD13543 compl (11) GlycosylationdTDP-glucose-4,6- OrfH 2712-3710 LanH (71/82) AAD13546 dehydratase compl(12) NDP-hexose-3- OrfQ 1391-2701 UrdQ (83/91) AAF72550 dehydratasecompl (13) NDP-hexose-2,3- OrfS* -561 compl LanS (68/74) AAD13549dehydratase (14) 4-ketoreductase OrfR 580-1335 compl LanR (66/77)AAD13548 (15) O-acyltransferase OrfY 5494-6687 compl MegY (42/58)AAG13909 (16) Regulation regulation OrfR1* 18603-compl (17) JadR1(58/70) AAB36584 Resistance transporter OrfJ 6780-8051 compl UrdJ2(51/61) AAF00207 (18) Unclear unknown Orf1 17692-18492 S. fradiaeAAD40806 (19) ORF12 (41/49) homologous to Orf2 3793-4848 S. coelicolorCAB72221 oxidoreductases compl (20) SCE56.02 (40/55)*Partial sequence compl = complementary sequence1.5 Expression Cloning

The two 9.5 kb PstI fragments were cloned into the plasmid pIJ486, anddesignated as pS11P2 and pS11P23. The plasmids were introduced into theS. lividans strain TK24, isolated from it and introduced further to S.argillaceus and then first into S. galilaeus mutant H039, and then intoS. galilaeus mutant H075.

EXAMPLE 2 Compounds Generated by 11P2 and 11P23 Clusters

2.1 Cultivation and Purification

According to the initial HPLC-DAD analysis, the strains TK24/pS11P2,HO39/pS11P2 and S. argillaceus/pS11P2 produced unknown compounds,together with known compounds related to corresponding parent strains.Each strain was fermented at 10 l scale for purification andidentification of unknown products. After seven days' fermentation (Elmedium, 28° C., 300 rpm, aeration 10 l/min) the mycelia were separatedwith ultrafiltration. Prior to the separation the pH of the broth wasadjusted to be between 4 and 5. The mycelia were extracted three timeswith methanol (3×1l). The supernatant was treated for 30 min with 300 gof Amberlite XAD-7 resin, which was collected and subsequently extractedwith 2 1 of methanol. Combined methanol extracts werevacuum-concentrated to 200 ml.

The liquid residue was loaded onto a RP-18 flash column (5×6 cm) andeluted with a descending gradient of methanol/water, starting from 70%of water. Fractions were analysed by TLC and pooled based on analysis.Pooled fractions were extracted with chloroform, washed with water andconcentrated to dryness. The dry residue was loaded onto a SiO₂ flashcolumn (2×10 cm) loaded with dichloromethane. The column was developedby increasing stepwise the portion of methanol in dichloromethane up to25%. Fractions were detected with TLC and pooled according to analysis.Pooled fractions were evaporated to dryness and applied to preparativeHPLC (RP-18, 250×10) using a descending gradient of acetonitrile-0.1%HCOOH eluent. Pooled pure fractions were extracted with chloroform anddried for spectroscopic evaluation. The production levels of theproducts (1-6, FIG. 1) in corresponding strains were below 10 mg/l.

2.2 Identification

Identification of the compounds was based on unambiguous assignation ofcarbon and proton resonances using a standard combination of HMBC, HSQC,TOCSY and NOESY experiments. The results are depicted in Tables 2 to 4below. The results were also confirmed with mass spectrometric (MS) datafrom compounds (1)-(3), giving the correct molecular mass for each, andexpected degradation patterns consistent with the structures. Thestructures of the compounds (4)-(6) were deduced from NMR-data.

MS Results for Compounds (1)-(3):

(1) EIMS, m/z (relative intensity): 338(M⁺/10), 320(35), 310(45),295(15), 280(100)

(2) EIMS, m/z (relative intensity): 368(M⁺/15), 350(100), 310(25),279(15)

(3) EIMS, m/z (relative intensity): 354(M⁺/5), 336(100), 326(7), 311(7),296(10) TABLE 2 ¹³C data (δ, multiplicity) for compounds (1)-(4) at 100MHz in CDCl₃ (1)-(2) and in 1:1 mixture of CDCl₃ and d₆-DMSO (3)-(4).9-OMe- 5-OH- 11-OH- rabelomycin rabelomycin rabelomycin rabelomycin Site(1) (2) (3) (4)  1 196.6(s) 196.0(s) 196.5(s) 196.6(s)  2  54.1(t) 53.1(t)  53.2(t)  51.9(t)  3  70.9(s)  71.0(s)  70.9(s)  71.1(s)  4 36.9(t)  43.0(t)  37.8(t)  38.2(t)  4a 150.6(s) 151.2(s) 135.7(s)150.0(s)  5 122.2(d) 121.0(d) 147.8(s) 120.6(d)  6 160.2(s) 162.1(s)150.6(s) 161.6(s)  6a 115.9(s) 116.6(s) 115.9(s) 115.2(s)  7 192.2(s)192.8(s) 192.4(s) 192.2(s)  7a 115.4(s) 115.4(s) 115.2(s) 115.0(s)  8160.9(s) 151.2(s) 161.1(s) 161.0(s)  9 120.6(d) 153.0(s) 122.9(d)120.8(d) 10 135.8(d) 117.8(d) 137.5(d) 119.6(d) 11 119.0(d) 120.2(d)118.8(d) 160.3(s) 11a 135.2(s) 126.2(s) 135.8(s) 116.8(s) 12 181.8(s)181.8(s) 181.5(s) 188.6(s) 12a 129.2(s) 137.0(s) 126.2(s) 125.9(s) 12b132.0(s) 129.9(s) 130.7(s) 130.0(s) 13  29.4(q)  29.0(q)  29.1(q) 28.9(q)  9-OMe —  55.2 — —

TABLE 3 ¹H data (δ, multiplicity, J_(hh), area) for compounds (1)-(4) at400 MHz in CDCl_(3 (1)-(2) and in 1:1 mixture of) CDCl₃ and d₆-DMSO(3)-(4). 9-OMe- 5-OH- 11-OH- rabelomycin rabelomycin rabelomycinrabelomycin Site (1) (2) (3) (4)  2a 3.01, d, 15.1, 1H 2.85, d, 14.4, 1H2.92, d, 13.6, 1H 3.00, d, 15.0, 1H  2b 2.95, d, 15.2, 1H 2.75, d, 14.4,1H 2.74, dd, 13.6, 2.93, d, 15.1, 1H 1.2, 1H  3-OH exchange exchangeexchange exchange  4a 3.08, brs, 2H 2.98, brs, 2H 3.15, dd, 17.6, 3.01,brs, 2H 1.2, 1H  4b — — 2.83, d, 17.6, 1H —  5 6.99, s, 1H 6.94, s, 1H —6.98, s, 1H  5-OH — — exchange —  6-OH 12.22, s, 1H 12.00, brs, 1Hexchange 12.47, s, 1H  8-OH 11.65, s, 1H 11.96, brs, 1H exchange 11.95,s, 1H  9 7.26, d, 7.6, 1H — 7.26, dd, 8.2, 7.25, d, 9.3, 1H 1.5, 1H 9-OMe — 3.91, s, 3H — — 10 7.65, dd, 8.1, 7.22, d, 8.0, 1H 7.74, dd,8.2, 7.20, d, 9.3, 1H 7.6, 1H 7.6, 1H 11 7.25, d, 8.1, 1H 7.52, d, 8.0,1H 7.51, dd, 7.6, — 1.4, 1H 11-OH — — — 12.16, s, 1H 13 1.49, s, 3H1.31, s, 3H 1.37, s, 3H 1.44, s, 3H

TABLE 4 ¹H (δ, multiplicity, J_(hh), area) and ¹³C (δ, multiplicity)spectral data for compounds (5) and (6) at 400 and 100 MHz,respectively, in CDCl₃ (5) and in d₆-DMSO (6). 19-methyl-SEK15 (5)ε-rhodomycinone (6) Site ¹³C ¹H ¹³C ¹H  1 163.5(s) — 119.7(d) 7.75, d,8.3, 1H  1-OH — 11.55, brs, 1H — —  2  88.2(d) 5.13, d, 1.9, 1H 137.2(d)7.61, dd, 8.3, 7.7, 1H  3 170.2(s) — 124.9(d) 7.22, d, 7.7, 1H  4101.1(d) 5.68, d, 2.0, 1H 162.9(s) —  4-OH — — — 11.95, s, 1H  4a — —115.9(s) —  5 163.7(s) — 190.8(s) —  5a — — 111.2(s) —  6  36.4(t) 3.57,s, 2H 155.9(s) —  6-OH — — — 13.33, s, 1H  6a — — 137.5(s) —  7 132.6(s)—  62.6(d) 5.25, brs, 1H  8 121.0(d) 6.75, dd, 8.1, 1.0, 1H  34.4(t)2.19, cm, 2H  9 130.1(d) 7.21, dd, 8.1, 7.8, 1H  71.4(s) — 10 114.6(d)6.78, dd, 7.8, 1.1, 1H  51.5(d) 4.18, s, 1H 10a — — 135.0(s) — 11153.8(s) — 157.0(s) — 11a — — 111.4(s) — 11-OH — 9.78, s, 1H — 12.77, s,1H 12 130.8(s) — 186.0(s) — 12a — — 133.3(s) — 13A 199.9(s) —  32.6(t)1.71, dq, 14.3, 6.3, 1H 13B — — — 1.45, dq, 14.3, 6.2, 1H 14 115.6(s) — 6.8(q) 1.08, t, 6.3, 3H 15 165.1(s) — 171.3(s) — 15-OH — 12.67, s, 1H —— 16 100.7(d) 6.12, d, 8.3, 1H  52.4(q) 3.65, s, 3H 17 163.2(s) — — —17-OH — 10.41, brs, 1H — — 18 111.6(d) 6.08, d, 8.3, 1H — — 19 143.0(s)— — — 20  21.5(q) 1.83, s, 3H — —Deposited Microorganisms

The following microorganisms were deposited in Deutsche Sammlung vonMikro-organismen und Zellkulturen (DSMZ), Mascheroder Weg 1 b, D-38124Braunschweig, Germany. Accession Microorganism number Deposition dateStreptomyces lividans TK24/pS11P2 DSM 14172 13 Mar. 2001 Streptomyceslividans TK24/pS11P23 DSM 14173 13 Mar. 2001Sequence Listing Free Text

For:

-   SEQ ID NO:2 “translate of OrfA, putative function: ketosynthase I”-   SEQ ID NO:3 “translate of OrfB, putative function: ketosynthase II”-   SEQ ID NO:4 “translate of OrfC, putative function: acyl carrier    protein”-   SEQ ID NO:5 “translate of OrfD, putative function: ketoreductase”-   SEQ ID NO:6 “translate of OrfE, putative function: oxygenase II”-   SEQ ID NO:7 “translate of OrfF, putative function: cyclase”-   SEQ ID NO:8 “translate of OrfL, putative function: cyclase”-   SEQ ID NO:9 “translate of OrfM, putative function: oxygenase I”-   SEQ ID NO: 10 “translate of OrfV, putative function: reductase I”-   SEQ ID NO: 11 “translate of OrfO, putative function: reductase II”-   SEQ ID NO: 12 “translate of OrfH, putative function:    dTDP-glucose-4,6-dehydratase”-   SEQ ID NO: 13 “translate of OrfQ, putative function:    NDP-hexose-3-dehydratase”-   SEQ ID NO: 14 “translate of OrfS, putative function:    NDP-hexose-2,3-dehydratase”-   SEQ ID NO: 15 “translate of OrfR, putative function:    4-ketoreductase”-   SEQ ID NO: 16 “translate of OrfY, putative function:    O-acyltransferase”-   SEQ ID NO: 17 “translate of OrfR1, putative function: regulation”-   SEQ ID NO: 18 “translate of OrfJ, putative function: transporter”-   SEQ ID NO:19 “translate of Orf1, putative function: unknown”-   SEQ ID NO:20 “translate of Orf2, putative function: oxidoreductase”-   SEQ ID NO:21 Description of Artificial Sequence: oligonucleotide    primer-   SEQ ID NO:22 Description of Artificial Sequence: oligonucleotide    primer    References-   Dairi, T., Hamano, Y., Furumai, T. and Oki, T. (1999). Development    of a self-cloning system for Actinomadura verrucosospora and    identification of polyketide synthase genes essential for production    of the angucyclic antibiotic pradimicin. Appl. Environ. Microbiol.    65:2703-2709.-   Decker, H. and Haag, S. (1995). Cloning and characterization of a    polyketide synthase gene from Streptomyces fradiae Tü2717, which    carries the genes for biosynthesis of the angucycline antibiotic    urdamycin A and a gene probably involved in its oxygenation. J.    Bacteriol. 177:6126-6136.-   Gould, J., Hong, S. and Carney, J. (1998). Cloning and heterologous    expression of genes from the kinamycin biosynthetic pathway of    Streptomyces murayamaensis. J. Antibiot. (Tokyo) 51:52-57.-   Han, L., Yang, K., Ramalingam, E., Mosher, R. and Vining, L. (1994).    Cloning and characterization of polyketide synthase genes for    jadomycin B biosynthesis in Streptomyces venezulae ISP5230.    Microbiology 140:3379-3389.-   Hong, S., Carney, J. and Bould, S. (1997). Cloning and heterologous    expression of the entire gene clusters for PD 116740 from    Streptomyces strain WP 4669 and tetrangulol and tetrangomycin from    Streptomyces rimosus NRRL 3016. J. Bacteriol. 179:470-476.-   Hopwood, D., Bibb, M., Chater, K., Keiser, T., Bruton, C., Kieser,    H., Lydiate, D., Smith, C., Ward, J., and Schrempf, H. (1985).    Genetic manipulation of Streptomyces: a laboratory manual. The John    Innes Foundation, Norwich, United Kingdom.-   Krohn, K. and Rohr, J. (1997) Angucyclines: total syntheses, new    structures, and biosynthetic studies of an emerging new class of    antibiotics. Top. Curr. Chem. 188:127-195.-   Metsä-Ketelä, M., Salo, V., Halo, L., Hautala, A., Hakala, J.,    Mäntsälä, P. and Ylihonko, K. (1999). An efficient approach for    screening minimal PKS genes from Streptomyces. FEMS Microbiol Lett.    180:1-6.-   Oki, Toshikazu, Dairi and Tohru, WO 98/11230. Polyketide synthases    of Actinomadura involved in pradimicin biosynthesis and the genes    encoding them. (Bristol-Myers Squibb Company, USA).-   Rohr, J. and Thiericke, R. (1992). Angucycline group antibiotics.    Nat. Prod Rep., 9:103-137.-   Sambrook, J., Fritsch, E. and Maniatis, T. (1989). Molecular    cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory    Press, Cold Spring Harbor, N.Y.-   Ward, J. M., Janssen, G. R., Kieser, T., Bibb, M. J., Buttner, M. J.    and Bibb, M. J. (1986). Construction and characterization of a    series of multicopy promoter-probe plasmid vectors for Streptomyces    using the aminoglycoside phosphotransferase from Tn5 as indicator.    Mol. Gen. Genet. 203:468-478.-   Westrich, L., Domann, S., Faust, B., Bedford, D., Hopwood, D. A. and    Bechthold, A. (1999). Cloning and characterization of a gene cluster    from Streptomyces cyanogenus S136 probably involved in landomycin    biosynthesis. FEMS Microbiol. Lett. 170:381-387-   Wright, F. and Bibb, M. (1992). Codon usage in the G+C-rich    Streptomyces genome. Gene. 113:55-65.-   Ylihonko, K., Hakala, J., Niemi, J., Lundell, J. and Mäntsalä, P.    (1994). Isolation and characterization of aclacinomycin    A-nonproducing Streptomyces galilaeus (ATCC 31615) mutants.    Microbiol. 140:1359-1365.

1. An isolated and purified DNA fragment, which is the gene cluster forrabelomycin biosynthetic pathway of Streptomyces bacteria, beingincluded in two 9.5 kb flanked PstI fragments of Streptomyces sp.genome.
 2. The DNA fragment according to claim 1, comprising thenucleotide sequence given in SEQ ID NO:1, or a sequence showing at least90% homology to said sequence.
 3. A recombinant DNA, which comprises theDNA fragment according to claim 1, or any one of the two 9.5 kb PstIfragments thereof as defined in claim 1, cloned in a plasmid replicatingin Streptomyces.
 4. The recombinant DNA according to claim 3, which isthe plasmid pS11P2, deposited in S. lividans strain TK24/pS11P2 with theaccession number DSM
 14172. 5. The recombinant DNA according to claim 3,which is the plasmid pS11P23, deposited in S. lividans strainTK24/pS11P23 with the accession number DSM
 14173. 6. A process for theproduction of hybrid polyketide compounds, comprising transferring theDNA fragment according to claim 1 into a Streptomyces host, cultivatingthe recombinant strain obtained, and isolating the compounds produced.7. The process according to claim 6, wherein the Streptomyces host is aStreptomyces lividans host.
 8. The process according to claim 6, whereinthe Streptomyces host is a Streptomyces argillaceus host.
 9. The processaccording to claim 6, wherein the Streptomyces host is a Streptomycesgalilaeus host.
 10. The process according to claim 6, wherein anangucycline is produced, which has the following formula (2)


11. The process according to claim 6, wherein an angucycline isproduced, which has the following formula (4)


12. A process for the production of hybrid polyketide compounds,comprising transferring at least one of the genes selected from thegroup consisting of Orfs A, B, C, D, E, F, L, M, V, O, H, Q, R, Y, J, 1and 2 into a Streptomyces host, said genes being derived from the DNAfragment according to claim 1, cultivating the recombinant strainobtained, and isolating the compounds produced.
 13. A process accordingto claim 12 for generating novel compounds for drug screening.
 14. Anangucycline compound, 9-OMe-rabelomycin, which has the following formula(2)


15. An angucycline compound, 11-OH-rabelomycin, which has the followingformula (4)